Hemogenic Endothelium Research Articles

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

P53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored.To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay.We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs.We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control.Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. DisclosuresKanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E11.5, and then migrate to the fetal liver after E12.5 for expansion. After E17.5, HSCs migrate to the bone marrow and reside in the bone marrow for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we are using cutting-edge spatial transcriptomics to comprehensively study the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and in search of the niche cells and factors for HSC expansion.To understand the spatial distribution and interactions between HSCs and niche cells in the fetal liver, we introduced 2 spatial transcriptomic methods, slide-seq, and 10x Visium, in our study on E14.5 mouse fetal liver. By integrating with a parallel single-cell sequencing analysis, we revealed the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 mouse fetal liver. Interestingly, we found that the PMCs were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs, indicating a supportive role of N-cadherin-expressing PMCs in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGF1, IGF2, TGFβ2, TGFβ3, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding was consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. Moreover, CPDB analysis indicated that other potential niche cells, such as endothelium cells, macrophages, and megakaryocytes, may support HSCs in different signal transduction pathways. For example, endothelium cells have an enriched expression of KITL, IGF2, DLL1, TGFβ1, and TGFβ2; macrophages have enriched expression of KITL, IFNγ, and TGFβ1; megakaryocytes have enriched expression of PF4, JAG2 and TGFβ1. Intriguingly, our previous studies showed that megakaryocytes could promote HSC expansion under stress conditions in the bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cad CreER;Cxcl12 and an N-cad CreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, conditional knockout of Cxcxl12 in N-cadherin-expressing cells also resulted in a myeloid-biased differentiation. We postulate that the knockout of Cxcl12 or Scf in N-cadherin-expressing cells leads to the migration of HSCs towards other potential niche cells, such as macrophages and megakaryocytes, which may induce HSC expansion and biased differentiation.In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches, such as spatial transcriptomics and fluorescence in situ hybridization (FISH), to verify the distribution changes of HSCs in N-cad CreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch Ligand Jagged1 Is a Fetal Liver Niche Factor for the Function of Embryonic Hematopoietic Stem Cells

Embryonic hematopoietic stem cells (HSC) expand rapidly during development in the fetal liver. Notch1 is required for emergence of the definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium, and is essential for survival and function of HSCs in the fetal liver. The identity of the ligand and the ligand-presenting cell during hematopoietic development would provide valuable information of the Notch signaling mechanism in HSCs as well as the identity of key niche cells that drive the expansion and cell fate decisions of embryonic HSCs. In the present study, we have taken a comprehensive approach to determine the ligands and cells that initiate Notch signaling in the mouse fetal liver. To this end, we have performed single-cell analysis for all Notch signaling proteins and many known targets in E14.5 fetal HSCs and adult bone marrow HSCs as well as fetal liver endothelial cells. We determined that Jagged1 (Jag1) is highly expressed in both endothelial cells as well as in fetal HSCs but not in adult HSCs. We have performed conditional loss-of-function analysis of Jag1 in fetal endothelial cells as well as in fetal hematopoietic lineages, where both myeloid and megakaryocytic progenitors are shown to express high levels of Jag1. Our results indicate that while loss of endothelial Jag1 has severe effects in embryonic vascular development, loss of hematopoietic Jag1 allows for normal fetal morphology, yet severely impedes the functional ability of fetal liver HSCs to expand and differentiate. RNA-Sequencing analysis of long-term fetal HSCs in Jag1-mutant embryos (VavCreJag f/f) revealed reduced expression of Gata2, Mllt3, Hoxa7, Angpt1 and IL-12a genes in fetal HSCs, which are well-known regulators of self-renewal and expansion. Our findings indicate that Jag1 is an essential niche factor for development of HSCs in the fetal liver and for functional potential of fetal HSCs once in the bone marrow microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Induced Pluripotent Stem Cell-Derived Neutrophil Granulocytes - Flow Cytometry Analysis Reveals Distinct Subpopulations

Severe congenital neutropenia (SCN) comprises a spectrum of monogenic disorders characterized by impaired differentiation and function of neutrophil granulocytes. Since animal models often do not fully recapitulate human SCN phenotypes and primary bone marrow samples of patients are scarce, alternative strategies are desirable to study the genetic causes and mechanisms. Induced pluripotent stem cells (iPSCs) can be differentiated into neutrophil granulocytes, thereby presenting an excellent tool to study hematopoiesis and especially neutrophil differentiation in health and disease in vitro.Recently, neutrophil progenitors and mature neutrophils of murine and human bone marrow have been characterized by single-cell RNA sequencing, mass cytometry and flow cytometry and are now referred to as proNeu, preNeu, immature-Neu and mature-Neu, at least in part reflecting conventional morphological classification of myeloblasts, promyelocytes, myelocytes/metamyelocytes and band and segmented neutrophils, respectively.Here we developed a flow cytometry antibody panel and gating strategy, which robustly identified distinct myeloid subsets in iPS-derived neutrophils. We adopted a differentiation protocol, which consists of feeder- and serum-free differentiation of iPS cells by mesoderm induction and patterning, followed by lineage progression through hemogenic endothelium to hematopoietic progenitors and finally mature neutrophil granulocytes. Floating cells arise, which can be harvested continuously and analyzed by flow cytometry.Based on expression of cell surface molecules, we defined four subpopulations: After selecting for single, live, CD45 + and CD14 - cells, the different progenitor stages were first defined by their expression of CD117 and CD49d. CD117 midCD49d high cells were further stratified into SSC lowCD34 + cells and SSC highCD34 - cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2/preNeu), respectively. These cells progressed to CD117 -CD49d mid and were CD11b +CD101 +, which defines myelocytes/metamyelocytes (immature-Neus). CD117 -CD49d low cells were CD11b +CD101 + and expressed additionally CD16, resembling band/segmented neutrophils (mature-Neus). Additionally, these iPS-derived cells progressively expressed CD35, which is also a maturation marker of human myeloid cells in vivo. May-Grünwald-Giemsa staining of these four subpopulations (CD117 midCD49d highSSC lowCD34 +, CD117 mid CD49d highSSC highCD34 -, CD117 - CD49d midand CD117 - CD49d low) revealed homogenous populations of sorted cells, morphologically resembling myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively.Ongoing studies in our lab make use of this model to a) validate the functional significance of rare genetic variants and b) further assess transcriptomic and proteomic changes on a single cell level. Thus, we provide a promising tool to study neutrophil differentiation in health and disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems.

Background. In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. Aim of the study. To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. Methods. We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. Results. We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. Conclusions. This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells’ intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/β-catenin signaling.

Hapln1b, a central organizer of the ECM, modulates kit signaling to control developmental hematopoiesis in zebrafish

AbstractDuring early vertebrate development, hematopoietic stem and progenitor cells (HSPCs) are produced in hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand to the fetal liver and the caudal hematopoietic tissue, in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta must be degraded to enable HSPCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSPC specification has never been addressed. In this study, hapln1b, a key component of the ECM, was specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSPCs in the early embryo, showing that hapln1b is necessary, at the correct level, to specify HSPCs in the hemogenic endothelium. Furthermore, the expression of hapln1b was necessary to maintain the integrity of the ECM through its link domain. By combining functional analyses and computer modeling, we showed that kitlgb interacts with the ECM to specify HSPCs. The findings show that the ECM is an integral component of the microenvironment and mediates the cytokine signaling that is necessary for HSPC specification.

Murine AGM single-cell profiling identifies a continuum of hemogenic endothelium differentiation marked by ACE

AbstractIn vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

AbstractRUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1-null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, although zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multilineages, raising the possibility that HSCs can emerge without RUNX1. Here, using 3 new zebrafish runx1−/− lines, we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-green fluorescent protein (GFP)+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1−/−-specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1−/− embryos. Interestingly, genetic inactivation of both gata2b and its paralog gata2a did not affect hematopoiesis. However, knocking out runx1 and any 3 of the 4 alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1−/− mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.

Glutamine metabolism regulates endothelial to hematopoietic transition and hematopoietic lineage specification

During hematopoietic development, definitive hematopoietic cells are derived from hemogenic endothelial (HE) cells through a process known as endothelial to hematopoietic transition (EHT). During EHT, transitioning cells proliferate and undergo progressive changes in gene expression culminating in the new cell identity with corresponding changes in function, phenotype and morphology. However, the metabolic pathways fueling this transition remain unclear. We show here that glutamine is a crucial regulator of EHT and a rate limiting metabolite in the hematopoietic differentiation of HE cells. Intriguingly, different hematopoietic lineages require distinct derivatives of glutamine. While both derivatives, α-ketoglutarate and nucleotides, are required for early erythroid differentiation of HE during glutamine deprivation, lymphoid differentiation relies on α-ketoglutarate alone. Furthermore, treatment of HE cells with α-ketoglutarate in glutamine-free conditions pushes their differentiation towards lymphoid lineages both in vitro and in vivo, following transplantation into NSG mice. Thus, we report an essential role for glutamine metabolism during EHT, regulating both the emergence and the specification of hematopoietic cells through its various derivatives.

Multipotent progenitors and hematopoietic stem cells arise independently from hemogenic endothelium in the mouse embryo.

SUMMARYDuring embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties.

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay.

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called “pre-HSCs.” Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed “11a- eKLS” (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis

Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages.

KIT Is Required for Fetal Liver Hematopoiesis.

In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial-to-hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed in hematopoietic progenitors that arise via endoHT from yolk sac hemogenic endothelium to generate early, transient hematopoietic waves. However, it remains unclear whether KIT has essential roles in early hematopoiesis. Here, we have combined single-cell expression studies with the analysis of knockout mice to show that KIT is dispensable for yolk sac endoHT but required for transient definitive hematopoiesis in the fetal liver.

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification

ABSTRACT Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

A connexin/ifi30 pathway bridges HSCs with their niche to dampen oxidative stress

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.

De novo generation of macrophage from placenta-derived hemogenic endothelium.

Macrophages play pivotal roles in immunity, hematopoiesis, and tissue homeostasis. In mammals, macrophages have been shown to originate from yolk-sac-derived erythro-myeloid progenitors and aorta-gonad-mesonephros (AGM)-derived hematopoietic stem cells. However, whether macrophages can arise from other embryonic sites remains unclear. Here, using single-cell RNA sequencing, we profile the transcriptional landscape of mouse fetal placental hematopoiesis. We uncover and experimentally validate that a CD44+ subpopulation of placental endothelial cells (ECs) exhibits hemogenic potential. Importantly, lineage tracing using the newly generated Hoxa13 reporter line shows that Hoxa13-labeled ECs can produce placental macrophages, named Hofbauer cell (HBC)-like cells. Furthermore, we identify two subtypes of HBC-like cells, and cell-cell interaction analysis identifies their potential roles in angiogenesis and antigen presentation, separately. Our study provides a comprehensive understanding of placental hematopoiesis and highlights the placenta as a source of macrophages, which has important implications for both basic and translational research.

GATA2 Is Essential for Endothelial-to-Hematopoietic Transition but Does Not Affect Formation of Hemogenic Endothelium

The formation of blood cells from hemogenic endothelium (HE) is a key element of leading to establishment of the hematopoietic system during embryogenesis. It has become increasingly clear that HE represents a distinct lineage of vascular endothelium with a capacity to undergo endothelial-to-hematopoietic transition (EHT). However, the processes guiding HE specification and EHT are poorly understood. The GATA2 transcription factor is of particular interest since it is critical for development of the entire hematopoietic system including hematopoietic stem cells (HSCs) during embryogenesis. Although GATA2 knockout has been shown to abrogate formation of intraortic hematopoietic clusters, it is not clear whether GATA2 is required for HE formation, EHT per se, or if it controls HE specification and EHT in a non-cell autonomous manner. To study the function of GATA2 during hematopoietic development, we engineered a GATA2 knockout H1 human embryonic stem cell (hESC) carrying doxycycline (DOX)-inducible GATA2 transgene. We demonstrated that GATA2 deficiency dramatically impaired definitive and primitive hematopoiesis from hESCs. To define the stage of development affected by GATA2, we treated iG2-/- and iG2+/+ hESCs with DOX in stepwise fashion, and evaluated the stage of development at which GATA2 induction has the most profound effect on hematopoiesis. We found that DOX treatment has the most dramatic effect on production of CD43+ cells and CFC potential when performed on days 3-4 or 4-5 of differentiation. In contrast, DOX treatment on days 0-2 suppressed differentiation, while treatment on days 5-6 has little to no effect. Since formation of HE and EHT in our system occurs during days 4-5 of differentiation, i.e. when we see the most dramatic effect of DOX treatment, we concluded that GATA2 may be important for HE formation or EHT. Flow cytometric analysis revealed that formation of VE-cadherin+CD43-CD73- HE, including DLL4+ arterial HE subset, and non-HE was not affected in iG2-/- hESCs, or following GATA2 induction with DOX. Next, we isolated iG2-/- HE from day 4 of differentiation and assessed how DOX treatment affected blood formation. In the absence of DOX, iG2-/-HE produced very few blood cells, while DOX treatment restored blood formation from HE. No significant difference was found in proliferation and in apoptosis of hematopoietic progenitors following EHT, regardless of the presence of GATA2, implying the specific effect of GATA2 on EHT.To define molecular pathways which involved in EHT impairment in iG2-/- cells, we performed RNAseq analysis of HE in DOX- and DOX+ conditions. Gene set enrichment analysis (GSEA) detected significant upregulation of sets of genes responding to TGFβ stimulation in iG2-/- HE on day 1 of secondary culture without DOX as compared to DOX treated cells, thereby suggesting that upregulation of target genes of TGFβ signaling may contribute to EHT impairment in GATA2-deficient cells. Indeed, treatment of iG2-/- HE with TGFβ-inhibitor significantly increased EHT and CD43+ hematopoietic progenitor production with CFU-G and GM potential. Overall, our studies suggest that GATA2 is dispensable for specification of HE, but is essential for EHT and proper hematopoietic specification. In addition, inhibiting TGFβ signaling partially restored the GATA2-dependent EHT in the absence of GATA2. DisclosuresNo relevant conflicts of interest to declare.

Induction of Arterial Type of Hemogenic Endothelium from Human Pluripotent Stem Cells

De novo production of hematopoietic stem cells (HSCs) and lymphoid cells from in vitro expandable human cells, such as human pluripotent stem cells (hPSCs), represents a promising approach for modeling and treating hematologic disease and immunotherapies. While the feasibility of generating engraftable hematopoietic cells and T lymphoid cells from hPSCs has been demonstrated, further advancing these technologies to bedside requires developing clinically safe protocols that allow for scalable production of therapeutic cells in defined conditions without genetic modification. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. The lack of venous contribution to HSCs when considered with the shared requirements for Notch, VEGF and Hedgehog signaling in both arterial fate acquisition and HSC formation also indicates that arterial specification could be critical prerequisite to HSC formation. Recent demonstration that HE represents a distinct CD73- endothelial lineage (Choi et al., 2012; Ditadi et al., 2015) and that hematopoietic specification already occurred at the HE stage (Elcheva et al., 2014; Guibentif et al., 2017; Slukvin, 2016), raises the question whether acquisition of arterial identity per se is sufficient for HE to specify toward lymphoid cells and HSCs, or whether arterial specification and signaling creates the permissive environment that allows for definitive hematopoiesis from HE. Because prior studies demonstrated that arterial specification is controlled by DLL4 enhancer which contains ETS-binding sites, we engineered H1 human embryonic stem cells (hESC) carrying doxycycline (DOX)-inducible ETS1 and assessed the effect of ETS1 on establishing of arterial program. We demonstrated that ETS1 upregulation during the mesodermal stage of development enhances arterial specification from hPSCs differentiated in chemically defined conditions. The boost in arterial programming by ETS1 was associated with promotion of HE formation with DLL4+CXCR4+/- arterial phenotype. Arterial type HE was highly enriched in cells with T and B lymphoid potential and the capacity to produce red blood cells with high expression of BCL11a and adult b-globin. As determined by limiting dilution assay the frequency of T cell precursors in DLL4+ HE was almost 100 times higher as compared to DLL4- HE (1/17 vs 1/1801). In addition, we demonstrated that specifying arterial HE and enhancing definitive hematopoiesis with lymphoid potential could be achieved through modulation of MAPK/ERK pathways. Indirect ERK activation through inhibition of phosphatidylinositol 3-kinase (PI3K) of branch of VEGFR signaling using LY294002 enhanced production of DLL4+ arterial HE, including the DLL4+CXCR4+ fraction, while ERK inhibition with U0126 treatment almost completely abrogated formation of arterial DLL4+ HE. We also observed a direct correlation between the efficacy of definitive hematopoiesis and arterial specification. When ERK pathways were activated following HE specification, production of multipotent CD235a/CD41a-CD45+ hematopoietic progenitors and multipotential CFCs were dramatically increased, while ERK inhibition abrogated production of these cell types. In addition, T lymphoid potential was significantly (more than six-fold) increased in cultures treated with LY294002 and entirely abrogated in cultures treated U0126. Together, these findings demonstrated that promotion of arterial specification in cultures could provide a novel strategy that aids in lymphopoiesis and eventually production of HSCs from hPSCs. DisclosuresNo relevant conflicts of interest to declare.

Engineering of Induced Pluripotent Stem Cell-Derived Hemogenic Endothelium

Background: Autologous cellular therapy of hematological malignancies and primary immunodeficiencies has been limited by the inability to produce functional hematopoietic stem cells (HSCs) from patient-derived induced pluripotent stem cells (iPS). Previous approaches have failed to demonstrate multi-lineage potential or secondary bone marrow engraftment in murine hosts - the gold standards for demonstrating HSC self-renewal and differentiation. Building upon recent evidence that HSCs derive from the definitive hemogenic endothelium (HE) of the embryonic lateral plate mesoderm, we set out to engineer iPS-derived HSCs by directed differentiation, and to investigate potential applications for modeling genetic blood diseases in vitro .Methods: Following embryoid body formation, human iPS derived from bone marrow mesenchymal stem cells underwent stepwise, morphogen-directed differentiation into HE, followed by polycistronic lentiviral transduction with five transcription factors (RUNX1, ERG, LCOR, HOXA5, HOXA9) on day 3 of endothelial-to-hematopoietic transition (EHT) induction, to enable further conversion to HSC-like cells with lymphoid potential in vitro and known multi-lineage engraftment capacity in vivo . A candidate screen of 17 cytokines, epigenetic modifiers and small molecules aimed at inducing HE proliferation was conducted during EHT induction in a RUNX1c gene reporter assay. To model human disease, dermal fibroblast-derived iPS from a hypomorphic RAG2 Omenn syndrome patient were differentiated into HSCs, and cocultured with MS5 or OP9-DL1 stromal cells to induce B or T cell development. Fluorescence-activated cell sorting (FACS) was performed weekly for 6 weeks after initiating lymphoid differentiation.Results: In the candidate screen, IFN-γ enhanced RUNX1c reporter 2.4-fold during EHT, which was confirmed by flow cytometry showing increased hematopoietic progenitor cells (CD34+CD45+). Several compounds were suppressive to EHT, including IFN-α, which abrogated EHT enhancement when combined with IFN-γ. Despite 1 week of lymphoid-directed differentiation, cell populations derived from mutant RAG2 iPS cells showed a predominance of myeloid progenitors (CD38+CD45+CD45RA+), with only 5% of progenitors manifesting a lymphoid phenotype (CD10+CD34+CD45RA+). In contrast, wildtype iPS-derived HSCs produced CD19+IgM- B cell progenitors as well as mature IgM+ B cells (8% versus .06% of CD19+CD21+ B cells), which were comparable to cord blood controls; CD3+CD4+CD8+ T cells were also identified after 3 weeks. Ultimately, we determined that RAG2 -mutant iPS were unable to generate CD19+ B cells and arrested at the early T cell stage (CD5+CD4-CD8-).Conclusion: We determined that transient exposure to IFN-γ greatly improved the EHT of HE, which may bolster the typically low yield of iPSC-derived HSCs. In a lymphoid differentiation assay, wildtype iPS-derived HE were comparable to CB in the formation of mature and immature B/T cells, whereas RAG2 -derived HEs arrested at the pro-thymocyte stage. This finding suggests that our approach to creating engraftable HSCs holds significant potential for modeling hematopoietic disease, both in animal models and as a “disease-on-a-dish,” and for developing therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.